Method of relieving pain in cancer by injecting lysozyme free of proteinaceous impurities



3,282,782 Patented Nov. 1, 1956 3,282,782 METHOD OF RELIEVING PAIN INCANCER BY INJECTING LYSOZYME FREE OF PROTEIN- ACEOUS IMPURITIES EneaGiuseppe Scolari, Florence, Italy, assignor to Societa ProdottiAntihiotici, Milan, Italy N Drawing. Filed Apr. 2, 1962, Ser. No.184,597 Claims. (Cl. 167-65) The present invention relates to a methodof treating cancer so as to relieve the pain thereof, and moreparticularly to the use of an agent which is effective as an antalgic inrelieving the pain of patients suffering from cancer.

The pain and suffering of patients affected with carcinoma of alldifferent types is of course well known, and it is also well known thatin the terminal stage of such carcinoma the patient can no longer obtainrelief by the use of the known analgesics and narcotics. As a matter offact, such patients become dehydrated and undernourished due to theintolerable and persistent pains which prevent them from sleeping,eating or drinking. In terminal stages of such carcinomas the patientseven become addicted to narcotics, without obtaining any pain relieftherefrom.

It is accordingly a primary object of the present invention to provide anew agent which is effective in the relief of pain of patients sufferingfrom cancer.

It is another object of the present invention to provide a non-narcotic,non-habit forming agent which acts as an effective analgesic in therelief of the pain of patients affected by carcinoma.

It is yet another object of the present invention to provide a method ofproducing the pain relieving agent of the present invention in a form inwhich the same can be administered, without side effect, for the reliefof pain of a patient affected by carcinoma.

Other objects and advantages of the present invention will be apparentfrom a further reading of the specification and of the appended claims.

With the above and other objects in view, the present invention mainlycomprises as a method of relieving the pain of a patient suffering fromcancer the injection of such patient with lysozyme.

Lysozyme is a basic polypeptide of enzymatic nature, positively charged,discovered by Fleming in 1922. It is present in blood, tissues andexcretions such as tears, and in womens milk. In higher concentrationsit occurs in brain and adrenals. Lysozyme is known to have a high degreeantibacterial properties, and to build a physiological defense mechanismwhich acts as a true natural protection against bacterial and viralinvasions.

In accordance with this invention it has been discovered that theinjection of patients suffering from cancer, either intravenously orintramusclarly, brings about a high degree of relief of the pain, and inmany cases a complete and total disappearance of pain. This relief ofpain has been achieved in cases which could no longer benefit by the useof known analgesics and narcotics, i.e. patients who could no longerobtain relief by the use of their analgesic or narcotic treatment weregiven relief by the use of lysozyme in accordance with the presentinvention.

While the administration of lysozyme in accordance with the presentinvention cannot in any way cure or even reduce the carcinoma, it doesbring a long lasting relief to the patient suffering from the carcinomaso as to at least give such patient a ray of hope and ease the sufferingof the patient.

In its pure form lysozyme is an impalpable white powder, ofsweetishtaste. Its structure has been studied by numerous workers, andrecently I. Jolles and P. Jolles have published a note concerning thecomplete developed formula of egg white lysozyme (Comptes Rendus desSeances de lAccademie des Sciences, vol. 253, pages 2773-75, Seance ofDecember 4, 1961).

Aqueous lysozyme solutions, when sterile, indefinitely maintaining theirchemical-physical and pharmacobiological characteristics. However,lysozyme in its normal form if used for the preparation of solutions forparenteral administration can cause disagreeable secondary phenomena. Itis therefore still another object of the present invention to provide amethod of purifying lysozyme so as to make the same suitable forparenteral administration without any danger of secondary side effects.

In accordance with the present invention it is possible to prepareampoules of lysozyme for intramuscular or intravenous injection. It isalso possible to prepare lysozyme in lyophilized form for dissolution ina retardsolvent solution, such as an aqueous polyvinylpyrrolidonesolution, for intramuscular or intravenous administration with theretard-solvent slowing the effect of the lysozyme and thereby giving ita more prolonged action.

Lysozyme was administered to numerous patients suffering from cancer ofvarious localizations in advanced stage of evolution, with numerousmetastases, in accordance with the present invention, the patients nolonger being surgically or radiologically recoverable, and sufferingterribly with persistent and intermittent pains which were refractory toevery other therapy. Among the various conditions from which thepatients were suffering were epithelial cancers of the oral cavity, lipsand cheek mucosa, cheeks, alveolar fornices, oral floor and palate,tonsils, and tongue. The great majority showed necrosis and ulcerations,and all but one had lymph glands metastases in the suprahyoid, milohyoidand juglar regions In one case only lymph glands metastases were presentin the sub-mandibular and latero-cervical regions, deriving from anepithelioma of the tongue, which was clinically healed underradium-therapy. In two of the patients there were also radiologicallyvertified metastases, respectively in the cervical and lumbar segment ofthe vertebral column.

There were also cases of endopelvic tumors, originating from theprostate, the recto-vaginal septum the uterus itself, with invasivegrowth into the parametria or/and into the vagina. In the two lattercases and in the case of the prostatic tumor metastases in the cervical,lumbar and sacral segments of the vertebral column were present. Alsoone case of epithelioma of the left maxillar and ethmoidal sinuses andone case of spinocellular epithelioma of the skin of the face, withdiffuse regional metastases in the lymph glands. In addition, a veryserious case in preagonic conditions, a Woman, operated 2 years beforefor mammary carcinoma, who suffered presently from numerous vertebralmetastases in the cervical and lumbosacral segments of the column. Alsotreated was a case of gastric cancer.

All the above mentioned patients were selected from cases that could notbenefit any more by surgical or radiological treatment; they had allsevere pains, either continuous or subcontinuous, with attacks oftormenting exacerbations at various frequencies. Most of these patientswere obliged to lay motionless in bed in coapted position; they wereunderfed and almost continuously sleepless; some of them were already inprecachectic conditions. In a few cases, only the pains were supportableand intermittent or of a dull type. The irradiations of the pains werethe customary ones for the single localizations. The patients with themost severe pains had been previously submitted to analgesic treatment,with morphine and morphinan derivatives or meperidine, sufferingtherefore of the connected intoxications; all other patients weretreated with minor analgesics or complementary drugs, as barbiturates,antipyretic analgesics, brome derivatives, ganglion-blocking agents andso on.

Lysozyme therapy was adopted after discontinuance of all other analgesictreatments; only thereapeutic integrations as analeptics, infusions andother not analgestic means were eventually used.

Lysozyme was administered by intramuscular injection, first at doses of75 mg. twice or three times daily and in the most resistant cases atdoses of 125-250 mg. twice daily. Lyophylized preparations orpreparations in a retarding solvent (polyvinylpyrrolidone) were used.With 20 cases treated the obtained results are briefly as follows: in 14cases a progressive diminution of pains up to complete or nearlycomplete disappearance; in 4 cases a remarkable reduction of pains withrestoration of the possibility of eating, resting overnight, getting upand moving (in any case a far improved condition than that previouslyobtained with opiates or their derivatives); in 2 cases a very slightamelioration or no amelioration at all.

An isolated attack of pain was sometimes recorded, even in favorablecases. However these attacks were far less severe than those whichafllicted the patients before the beginning of treatment and of ashorter duration. The amelioration lasted even after discontinuance oflysozyme treatment, eventually with the subsidiary use of harmlessanalgesic drugs.

In another 30 cases only intravenous injections were used. Lysozymetreatment was started after previous suspension of every other antalgictherapy. The lysozyme average posology, by intramuscular administrationin the cases previously mentioned was 125 mg. or 150 mg. doses(lyophilized lysozyme or lysozyme in a retard-solvent solution ofpolyvinylpyyrolidone) twice a day, during a period of 20-25 days.

In the more refractory cases it was necessary to apply to 2 dailyinjections of 250 mg. lysozyme. The average posology, intravenously inthe 30 cases herein discussed was of a daily injection of 250 mg. duringa period of 20-25 days.

Out of 20 cases treated with lysozyme intramuscularly there was obtainedthe following results:

Total or almost total disappearance of pain in 14 cases; a satisfactorysuffering decrease in 2 cases, and no effect in the other two cases.

Out of 30 cancerous patients affected by the so called refractory pains,in 20 cases, there was obtained by endovenous lysozyme administrationtotal disappearance of algic symptomatology; lysozyme antalgic actionwas partial in 7, and no effect in 3 cases. The percentage of positiveresults (total and partial disappearance of suffering) is 90%.

The best and most rapid results were reached by endovenousadministrations; within the first 24-36 hours of treatment animprovement in pain symptomatology was already evident; in the lessfavorable cases it was necessary to wait until the third to fifth day.Intramuscular administration has a more gradual effect (within 3-7 days)and positive results are reached only after lO-15 days of treatment.

A certain number of the cases treated by lysozyme endovenousadministration, concerns a group of patients previously submitted to alysozyme intramuscular treatment. When, owing to a continued worseningof the neoplastic process and the reappearance of painful symptomatologyby the renewal of the same treatment appeared inefficient, asatisfactory renewed sensibility to the therapy was obtained by usingendovenous administration.

In still another cases further treated with intramuscular lysozymeadministrations, concordant results were obtained. Based on thesesufficiently regular results. the following scheme of standard therapymay be applied:

(1) Immediate suspension of every other antalgic treatment.

(2) Initial therapy may be of a sole daily dosage of 250 mg. lysozymeintravenously. In case of a retarded reflect, after 3-5 days, redoublethe dose and administer it every day, endovenously, all at a time orfractionated, with an interval of 12 hours. (Lyophilized lysozyme, alsoin solution, at room temperature maintains unaltered for a long time.)

(3) When analgesic action appears stable for at least 20-25 days, it ispossible to try to reduce the doses to one-half, eventually usingintramuscular treatment. In positive cases, after 2-3 weeks, amaintaining therapy with a minimum dose of 75 mg., once or twice a daymay be tried. In fully favor-able cases, treatment can be suspended,eventually resorting to collateral drugs, with a tranquilizing,antistaminic and antispastic action.

(4) When painful symptomatology reappears (generally 25-40 days aftertreatment suspension) a new therapy cycle is required.

The above-mentioned therapeutic schema, can be naturally modifiedaccording to the most various particular circumstances.

It is clear from the treatments carried out using the lysozyme treatmentin accordance with the present invention that the lysozyme is effectiveto reduce pain in all types of cancer, and is not selectively effectivein only one or another type of cancer.

As indicated above it is another object of the present invention toprovide a method of producing lysozyme which can be used for injectionpurposes without the danger of undesired side effects.

With this object in view, the present invention mainly comprises theheating of an aqueous 2-10% lysozyme solution containing undesiredsecondary proteins to a temperature sufficiently high to denature thesecondary proteins, cooling the thus treated solution so as to causeprecipitation of the thus denatured secondary proteins, and filteringthe thus cooled solution so as to separate the denatured proteinstherefrom, thereby obtaining a lysozyme solution which can be used forinjection purposes.

The heating of the lysozyme solution is preferably carried out at atemperature of about 40-70 C. and at a pH value in the range of 3-7.This treatment results in the denaturation of the secondary proteinsthat are present in the form of traces, and the same are then removed byfiltering after the cooled solution, which is preferably cooled to atemperature of about 0-5 C. by being left in a refrigerator for 24hours, is removed from the refrigerator.

This heating method either taken alone, or in combination with adialysis method in which the lysozyme solution is dialyzed through acellophane membrane against distilled water results in the production ofa lysozyme solution which can be safely administered parenterally,either intramuscularly or intraveneously.

The following examples are given to illustrate the method of productionof the lysozyme solutions free of secondary side effects, the scope ofthe invention not, however, being limited by the details of theexamples.

EXAMPLE 1 An aqueous solution with lysozyme content of about 10%, andpH-values in the range of about 7 after a heat treatment at atemperature of 70 C. and cooling to 0 C. and filtering is placed in acellophane membrane against distilled water, and stirred for severalhours. The aqueous solutions containing the lysozyme passed through themembrane are put together and the lysozyme is separated by vacuumevaporation, or by salting, or finally, by flowing across car-boXylresin IRC 50, previously treated with a phosphate buffer 0.2 M and apH=7.18, and eluating then with a salt solution.

EXAMPLE 2 An aqueous solution with lysozyme contents of 2% and apH-value of 3, is heated at a temperature of 40 C. After cooling, thesolution is left overnight in a refrigerator, at a temperature in therange of 0-5 C. The

solution is then filtered, and the lysozyme separated as in the Example1.

Without further analysis, the foregoing will so fully reveal the gist ofthe present invention that others can by applying current knowledgereadily adapt it for various applications without omitting featuresthat, from the standpoint of prior art, fairly constitute essentialcharacteristics of the generic or specific aspects of this inventionand, therefore, such adaptations should and are intended to becomprehended within the meaning and range of equivalence of thefollowing claims.

What is claimed as new and desired to be secured by Letters Patent is:

1. Method of relieving the pain of a patient suffering from cancer,which comprises injecting such patient with lysozyme which is free ofproteinaceous impurities.

2. Method of relieving the pain of a patient suffering from cancer,which comprises injecting such patient with a sterile aqueous solutionof lysozyme which is free of proteinaceous impurities.

3. Method of relieving the pain of a patient suffering from cancer,which comprises injecting such patient with a sterile aqueousretard-solvent solution of lysozyme which is free of proteinaceousimpurit-ies.

4. Method of relieving the pain of a patient suffering from cancer,which comprises injecting such patient with a sterile aqueouspolyvinylpyrrolidone solution of lysozyme which is free of proteinaceousimpurities.

5. Method of relieving the pain of a patient suffering from cancer,which comprises intramuscularly injecting such patient with lysozymewhich has been previously freed of proteinaceous impurities.

6. Method of relieving the pain of a patient suffering from cancer,which comprises intravenously injecting such patient with lysozyme whichhas been previously freed of proteinaceous impurities.

7. Method of relieving the pain of a patient suffering from cancer,which comprises injecting such patient with lysozyme which is free ofproteinaceous impurities in a daily dose of 125 to 500 mg.

8. Method of relieving the pain of a patient suffering from cancer,which comprises parenterally injecting such patient with a sterileaqueous preparation of purified lysozyme, prepare-d by heating at about40-70 C. an impure aqueous 2-10% lysozyme-containing, proteinaceoussolution, having a pH value of 3-7, to denature undesired proteinaceousimpurities; cooling the solution at a temperature of about 0-5 C.;filtering the thus cooled solution so as to separate the denaturedproteins therefrom; and incorporating the purified lysozyme therebyobtained into an aqueous parenteral solution.

9. Method of relieving the pain of a patient suffering from cancer,which comprises parenterally injecting such patient with a sterileaqueous preparation of purified lysozyme, prepared by heating at about4070 C. an impure aqueous 2-10% lysozyme-containing, proteinaceoussolution, having a pH value of 3-7, to denature undesired proteinaceousimpurities; cooling the solution at a tem perature of about 0-5 C.;filtering the thus cooled solution so as to separate the denaturedproteins therefrom; dialyzing the aqueous solution containing thepurified lysozyme through a dialysis membrane against distilled water;and incorporating the purified lysozyme thereby obtained into an aqueousparenteral solution.

10. Method of relieving the pain of a patient suffering from cancer,which comprises parenterally injecting such patient with a sterileaqueous prolonged-action polyvinylpyrrolidone-containing parenteralsolution of purified lysozyme, prepared by heating at about 40-70" C. animpure aqueous 2-10% lysozyme-containing, proteinaceous solution, havinga pH value of 3-7, to denature undesired proteinaceous impurities;cooling the solution at a temperature of about 0-5 C.; filtering thethus cooled solution so as to separate the denatured proteins therefrom;dialyzing the aqueous solution containing the purified lysozyme througha dialysis membrane against distilled water; and incorporating thepurified lysozyme thereby obtained into an aqueous prolonged-actionpolyvinylpyrrolyidonecontaining parenteral solution.

References Cited by the Examiner UNITED STATES PATENTS 4/1951 Martin167--55 10/1957 Gustus 2602.1

OTHER REFERENCES Altucci et al., Lysozyme in Experimental VirusInfections, Giom. Mal. Infett. Parassit. II (12), pp. 1133- 1144, 1959(abstracted in English in Biological Abstracts, vol. 35, #71099-71103,1960).

Brancato, Action of Lysozyme on Peritoneal Adhesions, Arch. Ed. Atti.,Soc, Med. Chir. di Messina, 3, pp. 237-241 (1959); (abstracted inEnglish in Chem. Abstracts, 55 (No. 3), #2891F, February 6, 1961).

Calierio, Appearance of Granule-s in the Cytoplasm of Tumor-CellCultures in Contact With Ly-sozyme, Nature 184 (suppl. 4), pp. 202-203,1959.

DeDuve, Lysosomes and Chemotherapy, Biological Approaches to CancerChemotherapy, Harris, pp. 101- 112, Academic Press, NY. (1961), perBiological Abstracts 36 #69309 (1961).

Ferrari et al., Interaction Between Lysozyme and a Phospholipid ofNeoplastic Tissue (Oncolipin), Nature 192, p. 1187 (1961).

Matracia et al., Interaction of Phosphatides and Glycolipides of YoshidaAscites Sarcoma With Lysozyme, Boll, soc. Ital., Biol, sper. 37, pp.548-550 (1961); abstracted in English in Chem. Abstracts 55 (No. 25),#26218d, Dec. 11, 1961.

Meniga et al., Resolution of Some Protein Mixtures by Gradient ElutionPaper C-hromatograp-h, Anal. Biochem. (1), pp. 502-510 (1960);(abstracted in Engish in Chem. Abstracts 55 (No. 14), #13523 (B), July10, 1961).

=Runti, Structure and Pharmacological Activity of Lysozym-e, paperpresented at 1 st International Symposium on Lysomzyme of Fleming,Milan, April 3-5, 1959 (in Italian), 42 pages (Exhibit D, attached topaper No. 3, of record).

Sedati et al., Distribution of I-1-31 Labeled -Lysozy-me in Some Tissuesand Organ Fluids, Aggior-n Mal. Infez. 7, pp. 19-26 (1961); (abstractedin En-glished in Chem. Abstracts 55 (No. 25), #27643'B, Dec. 11, 1961).

LEWIS GOTTS, Primary Examiner.

MORRIS O. WOL'K, Examiner. S. K. ROSE, Assistant Examiner.

1. METHOD OF RELIVING THE PAIN OF A PATIENT SUFFERING FROM CANCER, WHICHCOMPRISES INJECTING SUCH PATIENT WITH LYSOZYME WHICH IS FREE OFPROTEINACEOUS IMPURITIES.